Male fertility requires continual spermatogenesis which relies on the actions of spermatogonial stem cells (SSCs). The SSC pool serves as a reservoir from which progenitor spermatogonia arise that transiently amplify in number before committing to differentiation in response to retinoic acid (RA). Maintenance of the germline requires that the SSC pool itself must be resistant to RA stimulation. The molecular signals that control SSC actions remain poorly understood. However, during the current project period we discovered that several molecules including multiple members of the helix-loop-helix (HLH) transcription factor family influence activities of SSCs and progenitor spermatogonia. In particular, we found that: 1) the HLH factor ID4 is expressed by SSCs specifically and promotes self-renewal; 2) the cell cycle regulator RB is required for maintenance of the SSC pool and physically interacts with ID4; 3) expression of the key stem cell transcription factor SOX2 is linked with the ID4+/SSC population; and 4) the class II HLH factors NEUROG3 and SOHLH1 are expressed by both SSC and progenitor spermatogonia but, in contrast to ID4, these molecules influence differentiation capacity of progenitors rather than self-renewal of SSCs. To facilitate our studies, we generated a novel mouse line in which ID4+ spermatogonia are marked by expression of GFP, and utilized this model to define features of the transcriptome that distinguish SSC and progenitor subtypes. The studies described in this renewal proposal are designed to extend these findings for advancing the understanding of mechanisms controlling maintenance of the SSC pool. Our aims are to: 1) define the mechanism regulating SSC proliferation by critically examining the importance of an ID4-RB interaction, 2) define the mechanism inducing an SSC-specific transcriptome by determining the importance of a link between ID4 and SOX2, and 3) determine the mechanism conferring resistance to RA signaling in SSCs by investigating whether ID4 antagonizes the ability of NEUROG3 and SOHLH1 to relay the RA response. The combined results from these studies will enhance our understanding of the biology of SSCs, and this knowledge will be useful in determining causes of male infertility and in the generation of new diagnostic and treatment tools. The knowledge gained may also aid in devising strategies to protect the SSC pool from detrimental effects of chemotherapy and radiation therapy that often eliminate the germline of male cancer patients, resulting in infertility. Moreover, spermatogenesis is a classic model of stem cell dependent lineages, thus knowledge gained about the biology of SSCs may be applicable to stem cells in other tissues.